Transplantation of organs, hematopoietic cells and somatic cells has been a crucial therapeutic regimen for patients suffering from a variety of maladies. Although the techniques necessary for transplants are quite straight-forward, the great stumbling block for successful transplantation has been the immune system. A fundamental problem has been the great vigor with which the host immune system reacts against introduction of antigens found in donor tissues or cells.
Transplantation of allogeneic donor (i.e., the same species but not genetically identical to the host patient) or xenogeneic donor (i.e., a species other than that of the host) grafts has posed particularly great difficulties. The continued functioning of any donor graft depends upon continued functioning of the donor cells that make up that graft. The cells of donor grafts, however, can elicit an immune reaction on the part of the host that, if unchecked, may lead to destruction of the graft.
One method of alleviating the reaction by the host against a graft has been administration of immunosuppressive treatment to the host. Unfortunately, despite the availability of new and very effective immunosuppressive drugs, recurrent episodes of acute and chronic graft rejection remain common, frequently causing loss of graft function. Moreover, the long-term success of transplantation is often limited by complications resulting from drug-related toxicity and from long-term immunosuppression (e.g. infections and secondary malignancies). In addition, transplantation of bone marrow cells (BMC) or small intestine, which are rich in immunocompetent lymphocytes, frequently is associated with a potential life-threatening complication due to graft versus host disease (GVHD).
It has been shown that a full hematopoietic chimera, i.e., a patient whose own BMC have been 100% replaced by permanently engrafted BMC from another individual (donor), can permanently accept donor-derived allografts with no need for maintenance immunosuppressive therapy. However, induction of full hematopoietic chimerism has been difficult to accomplish. First, substantially complete destruction of the host""s immunohematopoietic compartment (xe2x80x9clethalxe2x80x9d conditioning) is usually required for engraftment of matched and especially mismatched BMC. With lethal conditioning of the host, GVHD consistently causes morbidity or mortality. In such cases, T cell depletion of the graft hematopoietic material represents the only approach for effective prevention of GVHD. T cell depletion in turn is associated with an increased incidence of graft rejection. To overcome the problem of graft rejection, recipients of T cell depleted marrow allografts may require particularly strong conditioning or, alternatively, very high numbers of T cell depleted BMC. Subjecting patients to aggressive rejection-prevention protocols, such as total body irradiation (TBI) alone or TBI in combination with a short course of immunosuppressive drugs is unlikely to be accepted by clinicians treating patients in need of organ allografts.
It has been proposed that true bilateral tolerance associated with mixed donor/recipient hematopoietic chimerism, i.e., the condition in which a patient possesses both recipient (host) and donor hematopoietic stem cells, rather than with full chimerism, would be preferable in clinical organ transplantation. Several experimental protocols have been designed to induce transplantation tolerance leading to mixed chimerism. Conditioning has required the use of high dose TBI followed by infusion with a mixture of T cell depleted donor and recipient BMC (Sachs et al., Ann. Thorac. Surg., 56:1221 (1993); Ildstad et al., Nature, 307:168 (1984)) or inoculation with donor BMC after lower dose TBI and infusion of a mixture of antibodies against CD4+ T cells, CD8+ T cells and NK cells leading to general pancytopenia. Tomita et al., J. Immunol., 153:1087 (1994); Tomita et al., Transplantation, 61:469 (1996). An alternative approach has been developed recently involving irradiation with a sublethal dose of TBI and inoculation with a very high number of T cell depleted donor-derived hematopoietic cells. Reisner et al., Immunol. Today, 16:437 (1995); Bachar-Lustig et al., Nature Medicine, 12:1268 (1986). Tolerogenic treatments using cyclophosphamide (hereinafter also referred to as xe2x80x9cCytoxanxe2x80x9d or xe2x80x9cCyxe2x80x9d) in combination with TBI have also been described.
Total lymphoid irradiation (TLI) has been employed successfully as the sole preparatory regimen prior to infusion with donor BMC, to induce mixed hematopoietic chimerism and bilateral transplantation tolerance. Slavin S., Immunol. Today, 3:88 (1987); Slavin et al., Isr. J. Med. Sci., 22:264 (1986). TLI is non-myeloablative and routinely given safely on an outpatient basis to transplant recipients and patients with Hodgkin""s disease. Unfortunately, consistent induction of chimerism using TLI has required very high cumulative doses of radiation (3,400-4,400 cGy) that again would not be desirable for transplant recipients. TLI has significant advantages over TBI, especially in the clinical setting. TLI, which involves selective irradiation of the lymphoid compartment without exposing the whole body to ionizing irradiation, is well tolerated. In addition, TLI preserves intact a significant portion of the host""s immunohematopoietic system, with resultant retained memory to recall antigens including infective agents. However, long courses of TLI can be time consuming and may be associated with short and long-term side effects that may not be suitable for routine clinical application.
The invention provides a new method for treating a host mammal to induce transplantation tolerance to cell, tissue and organ allografts and xenografts. Such transplants can provide replacement therapy for enzyme or metabolic disorders and adoptive immunotherapy for cancer and life-threatening infections in humans. The method also can be used to provide new animal models for tolerance induction toward allogeneic and xenogeneic cells. The invention also provides a new method of non-syngeneic cell therapy in which the cell population used for therapy is substantially depleted of responsiveness to host antigens prior to administration to the host.
In general, the invention features a method of treating a host mammal, including (a) administering donor antigens from a non-syngeneic donor to the host mammal; (b) administering a non-myeloablative dose of lymphocytotoxic agent (e.g., cyclophosphamide) or tolerizing agent to the host mammal to selectively eliminate the host mammal""s lymphocytes responding to the donor antigens; and (c) administering a preparation of hematopoietic stem cells from the non-syngeneic donor to the host mammal.
Prior to step (a), the host mammal can be administered an immunosuppressive agent in a non-myeloablative regimen sufficient to decrease the host mammal""s functional T lymphocyte population. The immunosuppressive agent can include one or more of an immunosuppressive drug, an alkylating agent, ionizing radiation, or anti-leukocyte or anti-leukocyte function antibodies. It is particularly advantageous to use a short course of TLI (sTLI) as the immunosuppressive agent, for example 1-12, frequently 1-6, doses of 200 cGy/dose.
The donor antigens administered to the host mammal can include non-cellular antigens, cells, tissues and/or organs. For example, the donor antigens can include hematopoietic stem cells or other viable cells. If the donor antigens include viable cells such as hematopoietic stem cells, then the immunosuppressive regimen referenced above should decrease the T lymphocyte population of the host to a level permitting at least transient survival of the donor""s cells. For example the T lymphocyte population of the host can be decreased by 90%, 95% or 99%
The host mammal can be an animal or a human, for example a human cancer patient. The donor can be allogeneic or xenogeneic to the host mammal. Following performance of the method, the host mammal""s blood can contain 20% or more donor cells. After administering the preparation of donor hematopoietic stem cells, with resultant engraftment of such cells in the host, the host can be treated with allogeneic cell therapy. This involves infusing allogeneic lymphocytes from the donor into the host mammal. Alternatively, the host can receive transplanted cells, tissues or organs from the donor, with the transplants becoming engrafted in the host due to the donor-specific tolerance induced in the host mammal.
In another aspect, the invention features a host-derived hematopoietic cell composition, including host-originating and donor-originating hematopoietic cells, with the composition being depleted of donor-specific, host-originating lymphocytes. The hematopoietic cell composition can be made by treating a host mammal as described above, then isolating the hematopoietic cell composition from the host mammal.
In a further aspect, the invention features a method of making a non-human mammal/human chimera. This involves performing the methods described above, with the host mammal being a non-human mammal and the donor being a human being. The host mammal can be, for example, a rodent or pig. The result is a rodent, pig or other non-human mammal stably engrafted with human hematopoietic stem cells. As such, the non-human mammal host constitutes a hematopoietic mixed chimera.
The invention also encompasses a composition of cells containing a cell population from a first individual mammal. The cell population contains lymphocytes and is depleted of responsiveness to antigens of a second individual mammal that is non-syngeneic (i.e., allogeneic or xenogeneic) with the first individual mammal. The depletion of responsiveness is by a method involving the following sequential steps: (a) administering an antigen source expressed by the second individual mammal to the first individual mammal; (b) administering a non-myeloablative dose of a lymphocytotoxic or tolerizing agent to the first individual mammal; (c) administering a preparation of hemopoietic cells from the second individual mammal to the first individual mammal; and (d) isolating the cell population from the first individual mammal. In the composition of the invention, cells endogenous to the first individual mammal are 50% to 100% of the cells of the population. The antigen used can be cancer cells and the first individual mammal and the second individual mammal can both be humans. Alternatively, the first individual mammal can be a non-human primate, and said second individual mammal can be a human, or the first individual mammal can be a pig and said second individual mammal can a human.
The invention also features a method of treating a mammal with non-syngeneic cell therapy. The method involves infusing a population of cells from a donor mammal into a host mammal, with the donor mammal and the host mammal being non-syngeneic with each other. The cell population can contain lymphocytes, and prior to infusing, the cell population can be depleted of responsiveness to antigens expressed by the host mammal. The depletion of responsiveness can be by substantially eliminating T cells from the cell population. Elimination of T cells can be by exposing the cell population to an immunosuppressive agent in a non-myeloablative regimen or by contacting the cell with mafosphamide. These eliminations can be performed in vitro or in vivo.
Alternatively, the depletion of the cell population can be accomplished by contacting the lymphocyte population with a composition comprising antigens expressed by said host mammal and the contacting can be in vitro or by administering the antigens to the first individual mammal. The method can further include the step of, after the contacting with the antigen composition, delivering a non-myeloablative dose of a lymphocytotoxic or tolerizing agent to the lymphocyte population. This delivering can be in vitro or by administering the non-myeloablative dose to the donor mammal. The method can also optionally include the steps of: (a) after the delivering, administering a preparation of hemopoietic stem cells from the host mammal to the donor mammal; and/or (b) prior to the contacting with antigen, exposing the lymphocyte population to an immunosuppressive agent in a non-myeloablative regimen sufficient to decrease the number of functional T lymphocytes in the lymphocyte population. In (b) the exposing can be in vitro or by administering the immunosuppressive agent to the donor mammal. The antigen composition can contain one or more antigen sources, e.g., cells, organs, tissues, and non-cellular antigens. For example, the antigen can include hemopoietic cells or cancer cells expressing major histocompatibility complex molecules of the host mammal. The cancer cells can, for example, be from the host mammal.
Also within the invention is an article of manufacture that includes packaging material and a biological cell container within the packaging material. The cell container can contain a composition that includes hematopoietic stem cells and the packaging material can contain a label or package insert indicating that the hematopoietic stem cells are to be used in step (a) or step (c) in a method of inducing non-syngeneic donor-specific tolerance in a host mammal. The method includes the steps of: (a) administering donor antigens from a non-syngeneic donor to the host mammal; (b) administering a non-myeloablative dose of lymphocytotoxic or tolerizing agent to the host mammal to selectively eliminate the host mammal""s lymphocytes responding to the donor antigens; and (c) administering a preparation of hematopoietic stem cells from the non-syngeneic donor to the host mammal.
Another article of manufacture encompassed by the invention is one that includes packaging material, a biological cell container within said packaging material, with the cell container containing any of the cell compositions of the invention described above. The packaging material contains a label or package insert indicating that the composition is to be used in a method of treatment including administering the composition to a second individual mammal that is in need of the composition.
The invention also features a method of inducing tolerance in a host mammal to a graft from a non-syngeneic host mammal. The method includes the following steps: (a) administering donor antigens from a non-syngeneic donor to the host mammal; (b) administering an immunosuppressive agent to the host mammal in a non-myeloablative regimen sufficient to decrease the host mammal""s functional T lymphocyte population; (c) transplanting cells, a tissue, or an organ from the donor into the host animal, (d) administering a non-myeloablative dose of lymphocytotoxic or tolerizing agent to the host mammal to selectively eliminate the host mammal""s lymphocytes responding to the donor antigens; and (e) administering a preparation of hematopoietic stem cells from the non-syngeneic donor to the host mammal. Steps (a), (b), and (c) of the method are performed on the same day and prior to steps (d) and (e).
The term xe2x80x9cnon-myeloablativexe2x80x9d as used herein includes any therapy that does not eliminate substantially all hematopoietic cells of host origin. xe2x80x9cTransplantationxe2x80x9d as used herein refers to transplantation of any donor-derived material including cells, tissues and organs. The cells may be hematopoietic or non-hematopoietic. xe2x80x9cDonor antigensxe2x80x9d as used herein refers to any donor-derived material that elicits a host immune response, including non-cellular antigens, cells, tissues or organs. Stem cells are particularly useful as donor antigens. A xe2x80x9clymphocytotoxic agentxe2x80x9d is an agent that kills T cells or paralyzes T cell function. A xe2x80x9ctolerizing agentxe2x80x9d is an agent that energizes or xe2x80x9cvetosxe2x80x9d T cells by preventing development of normal T cell-dependent responses. The term xe2x80x9ccancerxe2x80x9d as used herein includes all pathological conditions involving malignant cells; this can include xe2x80x9csolidxe2x80x9d tumors arising in solid tissues or organs as well as hematopoietic tumors such as leukemias and lymphomas. The term xe2x80x9cdonor-specific tolerancexe2x80x9d as used herein refers to tolerance of the host to donor-derived material. xe2x80x9cNon-syngeneicxe2x80x9d as used herein can be allogeneic or xenogeneic. xe2x80x9cDepletion of responsivenessxe2x80x9d in a particular cell population, as used herein, means either a decrease in the number of responsive cells, a decrease in the responsiveness of responsive cells, or both. Where cells are herein said to be xe2x80x9cendogenousxe2x80x9d to an individual mammal, it is understood that the cells themselves, or their precursors, were in that individual mammal prior to any administration of cells from another individual mammal.
Induction of donor-specific tolerance across strong major histocompatibility complex MHC and minor histocompatibility loci (MiHL) barriers, as well as across species barriers (xenogeneic tolerance) may be achieved in mammalian hosts using the tolerogenic treatment described herein. Induction of donor-specific transplantation tolerance while avoiding the need for maintenance immunosuppressive treatment is a highly desirable goal in clinical transplantation.
The non-myeloablative tolerogenic treatment described herein induces a state of long-lasting donor-specific tolerance to a wide variety of donor-derived material. Such an approach is attractive for allogeneic and xenogeneic transplantation of cells, tissues and organs in clinical settings, since all the steps of the protocol are well tolerated and relatively safe. Since there is no need to eradicate the entire host immunohematopoietic system during the course of the procedure, the recipients retain immune memory and are in a better position to resist graft-versus-host disease on the one hand and infectious complications on the other. This can be of crucial importance in clinical practice. The protocols for inducing donor-specific tolerance may be delivered, at least in part, as outpatient procedures.
The methods of non-syngeneic cell therapy provided herein can be especially useful in conditions in which cell, tissue, or organ failure or misfunction occurs. They can therefore be useful in, for example, metabolic deficiencies (including genetic metabolic deficiencies), autoimmune diseases, and cancer. The methods are therefore useful in passively transferring, from a donor to a host, immunity to one more infectious agents. They can be used without prior treatment of the host or subsequent to tolerization of the host to donor antigens by one of the tolerization methods of the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.